Paired Non-small Cell Lung Cancer Tissues Research Articles

Fibre-based fluorescence-lifetime imaging microscopy: a real-time biopsy guidance tool for suspected lung cancer.

Lung cancer is the most common cause of cancer-related deaths worldwide. Early detection improves outcomes, however, existing sampling techniques are associated with suboptimal diagnostic yield and procedure-related complications. Autofluorescence-based fluorescence-lifetime imaging microscopy (FLIM), a technique which measures endogenous fluorophore decay rates, may aid identification of optimal biopsy sites in suspected lung cancer. Our fibre-based fluorescence-lifetime imaging system, utilising 488 nm excitation, which is deliverable via existing diagnostic platforms, enables real-time visualisation and lifetime analysis of distal alveolar lung structure. We evaluated the diagnostic accuracy of the fibre-based fluorescence-lifetime imaging system to detect changes in fluorescence lifetime in freshly resected ex vivo lung cancer and adjacent healthy tissue as a first step towards future translation. The study compares paired non-small cell lung cancer (NSCLC) and non-cancerous tissues with gold standard diagnostic pathology to assess the performance of the technique. Paired NSCLC and non-cancerous lung tissues were obtained from thoracic resection patients (N=21). A clinically compatible 488 nm fluorescence-lifetime endomicroscopy platform was used to acquire simultaneous fluorescence intensity and lifetime images. Fluorescence lifetimes were calculated using a computationally-lightweight, rapid lifetime determination method. Fluorescence lifetime was significantly reduced in ex vivo lung cancer, compared with non-cancerous lung tissue [mean ± standard deviation (SD), 1.79±0.40 vs. 2.15±0.26 ns, P<0.0001], and fluorescence intensity images demonstrated distortion of alveolar elastin autofluorescence structure. Fibre-based fluorescence-lifetime imaging demonstrated good performance characteristics for distinguishing lung cancer, from adjacent non-cancerous tissue, with 81.0% sensitivity and 71.4% specificity. Our novel fibre-based fluorescence-lifetime imaging system, which enables label-free imaging and quantitative lifetime analysis, discriminates ex vivo lung cancer from adjacent healthy tissue. This minimally invasive technique has potential to be translated as a real-time biopsy guidance tool, capable of optimising diagnostic accuracy in lung cancer.

Lnc AC016727.1/BACH1/HIF-1 α signal loop promotes the progression of non-small cell lung cancer

BackgroundLong noncoding RNAs (lncRNAs) have been reported to play vital roles in the development and progression of cancer. However, their biological significance and functional mechanisms in non-small cell lung cancer (NSCLC) are mostly unclear.MethodsWe performed RNA-sequencing to predict the differential expression of lncRNAs in clinical NSCLC and paired paracancerous lung tissues. To identify lncRNA expression, quantitative polymerase chain reaction (qPCR) was used. Using both cell and mouse models, We studied lncRNA AC016727.1’s function in NSCLC growth and metastasis. Western blot assays, dual luciferase reporter assays, and chromatin immunoprecipitation were used to analyze the functional mechanism of lncRNA AC016727.1.ResultsOur larger NSCLC cohorts validated that the lncRNA AC016727.1 was upregulated in 94 paired NSCLC tissues and correlated with poor survival. Functionally, lncRNA AC016727.1 downregulation inhibited NSCLC cell proliferation, aerobic glycolysis, EMT, and migration, inducing apoptosis. Conversely, upregulated lncRNA AC016727.1 expression exhibited the opposite effect, promoting NSCLC cell survival. Importantly, lncRNA AC016727.1 knockdown inhibited lung cancer growth and slowed the progression of lung metastasis in nude mouse models. Mechanistically, lncRNA AC016727.1 upregulated BACH1 target gene expression by acting as a sponge for miR-98-5p, thereby functioning as a competing endogenous RNA. The function of lncRNA AC016727.1 is mediated by the miR-98-5p/BACH1 axis in NSCLC cells. Meanwhile, the transcription factor HIF-1α can bind to the promoter and activate lncRNA AC016727.1 transcription. lncRNA AC016727.1 regulates HIF-1α expression via BACH1 in NSCLC and forms the lncRNA AC016727.1/BACH1/HIF-1α signaling loop under hypoxic conditions.ConclusionOur study reveals a novel lncRNA AC016727.1/BACH1/HIF-1α signaling loop in the progression of NSCLC under hypoxic conditions, suggesting that lncRNA AC016727.1 could act as a useful biomarker for NSCLC and a new therapeutic target.

LncRNA NBR2 Regulates Cancer Cell Stemness and Predicts Survival in Non-small Cell Cancer Patients by Downregulating TGF-β1.

LncRNA NBR2 is a key regulator in cancer metabolism. However, its role in lung cancer is unknown. This study aimed to explore the function of NBR2 in non-small cell lung cancer (NSCLC), which is the most common type of lung cancer. Paired NSCLC and non-cancer tissues were collected from 68 patients with NSCLC. The expression of NBR2 and transforming growth factor-β1 (TGF-β1) in these samples was analyzed by RT-qPCR. The prognostic value of NBR2 for NSCLC was explored by performing a 5-year follow-up study. The interaction between NBR2 and TGF-β1 in two NSCLC cell lines was detected by overexpression assay, followed by RT-qPCR and Western blot analysis. Flow cytometry was performed to evaluate the role of NBR2 and TGF-β1 in regulating NSCLC cell stemness. NBR2 was significantly downregulated in NSCLC tissues than that in non-cancer tissues of NSCLC patients, and low expression levels of NBR2 predicted poor survival. TGF-β1 was significantly upregulated in NSCLC tissues than that in non-cancer tissues, and was inversely correlated with NBR2. Overexpression of NBR2 downregulated TGF-β1, while overexpression of TGF-β1 did not affect the expression of NBR2. Overexpression of NBR2 inhibited, while overexpression of TGF-β1 promoted NSCLC cell stemness. Overexpression of TGF-β1 attenuated the effects of overexpression of NBR2. Mechanically, NBR2 interacted with Notch1 protein to inhibit its expression, thereby inhibiting the expression of TGF-β1 and further affecting the proportion of CD133+ cells. LncRNA NBR2 regulates cancer cell stemness and predicts survival in NSCLC possibly by downregulating TGF-β1 through Notch1.

LncRNA ELFN1-AS1 predicts poor prognosis and promotes tumor progression of non-small cell lung cancer by sponging miR-497.

More novel biomarkers need to be discovered to improve the therapeutic efficiency of non-small cell lung cancer (NSCLC). lncRNA ELFN1-AS1 (ELFN1-AS1) was proved to play crucial roles in numerous diseases, its intention in NSCLC remains unclear. This study aimed to investigate the function of ELFN1-AS1 and its potential mechanism in NSCLC development. A total of 117 NSCLC patients were recruited and provided paired NSCLC tissues and normal tissues. The expression of ELFN1-AS1 was analyzed by PCR. The biological function of ELFN1-AS1 was estimated by CCK8 and Transwell assay. Additionally, the potential mechanism underlying the function of ELFN1-AS1 was explored by the dual-luciferase reporter assay and western blotting. The significant upregulation of ELFN1-AS1 was found in NSCLC tissues and cells, which was closely associated with the TNM stage, lymph node metastasis status, and overall survival of patients. The knockdown of ELFN1-AS1 was found to inhibit the cellular processes and EMT of NSCLC. Moreover, ELFN1-AS1 was found to serve as a sponge to binding with miR-497, and CCNE1 was demonstrated to be the downstream target of miR-497, which was speculated as the potential mechanism underlying the function of ELFN1-AS1. ELFN1-AS1 acts as an independent prognostic biomarker and tumor promoter of NSCLC by sponging miR-497/CCNE1 axis.

Abstract LB549: LncRNA PCAT1 activates SOX2 and suppresses radioimmune responses via regulating cGAS/STING signaling in non-small cell lung cancer

Abstract Background: The long non-coding RNA (lncRNA) PCAT1 is upregulated in non-small cell lung cancer (NSCLC), and promotes tumor growth and metastasis, but its roles in the radioimmune responses remain unknown. We aimed to investigate the impacts of PCAT1 on tumorigenesis and radioimmune responses and the underlying molecular mechanisms in NSCLC. Methods: Comprehensive bioinformatics analysis was performed to identify immunosuppressive lncRNAs involved with tumor invasion in NSCLC. The expression levels of PCAT1 were analyzed by in situ hybridization in 55 paired NSCLC and adjacent normal tissues. Both loss- and gain-of-function assays were performed to evaluate the functions of PCAT1 and SOX2 in NSCLC cell behaviors in vivo and in vitro. Bioinformatic analyses, chromatin isolation by RNA purification and dual-luciferase reporter assays were applied to validate the regulatory effects of PCAT1 on SOX2 expression. Chromatin immunoprecipitation, luciferase, and rescue assays were utilized to identify the relationship between SOX2 and the cGAS/STING signaling. Results: PCAT1 was immunosuppressive and related with NSCLC invasion. Increased PCAT1 was negatively correlated with immune cell infiltration in NSCLC. PCAT1 knockdown restrained proliferation, increased apoptosis, and inhibited cell migration and invasion in vivo and in vitro. PCAT1 activated SOX2 that accelerated tumorigenesis and immunosuppression. SOX2 promoted tumor growth through inhibiting cytotoxic T cell immunity. Moreover, SOX2 restrained cGAS transcription and hampered downstream type I IFN-induced immune responses. Inhibition of PCAT1/SOX2 in collaboration with radiation further inhibited tumor growth, and activated the cGAS/STING signaling pathway, which enhanced the immune responses of radiotherapy in NSCLC. Conclusions: PCAT1/SOX2 axis promoted tumorigenesis and immunosuppression through inhibition of cGAS/STING signaling-mediated T-cell activation. Inhibition of PCAT1 and SOX2 synergized with radiotherapy to activate the immune response and could serve as potential therapeutic targets. Citation Format: Yanping Gao, Nannan Zhang, Yan Gong, Conghua Xie. LncRNA PCAT1 activates SOX2 and suppresses radioimmune responses via regulating cGAS/STING signaling in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB549.

LncRNA PCAT1 activates SOX2 and suppresses radioimmune responses via regulating cGAS/STING signalling in non-small cell lung cancer.

BackgroundThe expression of long non‐coding RNA (lncRNA) prostate cancer‐associated ncRNA transcripts 1 (PCAT1) is increased in non‐small cell lung cancer (NSCLC). It stimulates tumour growth and metastasis, but its role in the radioimmune responses remain unknown. We aimed to explore the impacts of PCAT1 on tumorigenesis and radioimmune responses and the underlying molecular mechanisms in NSCLC.MethodsComprehensive bioinformatics analysis was performed to identify immunosuppressive lncRNAs involved with tumour invasion in NSCLC. The expression levels of PCAT1 were analysed by in situ hybridisation in 55 paired NSCLC tissues and adjacent normal tissues. Both loss‐ and gain‐of‐function assays were performed to examine the effects of PCAT1 and SOX2 on NSCLC cell behaviours in vivo and in vitro. Bioinformatic analyses, chromatin isolation by RNA purification (ChIRP) and dual‐luciferase reporter assays were applied to validate the regulatory effects of PCAT1 on SOX2 expression. Chromatin immunoprecipitation, luciferase and rescue assays were utilised to identify the relationship between SOX2 and the cGAS/stimulator of interferon genes (STING) signalling.ResultsPCAT1 was immunosuppressive and related with NSCLC invasion. Increased PCAT1 was negatively correlated with immune cell infiltration in NSCLC. PCAT1 knockdown restrained proliferation, increased apoptosis, and repressed cell metastasis in vivo and in vitro. PCAT1 activated SOX2 that accelerated tumorigenesis and immunosuppression. SOX2 promoted tumour growth through inhibiting cytotoxic T‐cell immunity. Moreover, SOX2 restrained cGAS transcription and hampered downstream type I interferon (IFN)‐induced immune responses. Inhibition of PCAT1/SOX2 in collaboration with radiation further inhibited tumour growth, and initiated the cGAS/STING signalling pathway, which enhanced the immune responses of radiotherapy in NSCLC.ConclusionsPCAT1/SOX2 axis promoted tumorigenesis and immunosuppression through inhibition of cGAS/STING signalling‐mediated T‐cell activation. Inhibition of PCAT1 and SOX2 synergised with radiotherapy to activate the immune response and could serve as potential therapeutic targets.

Circular RNA circ-PLCD1 functions as a tumor suppressor in non-small cell lung cancer by inactivation of PI3K/AKT signaling pathway.

Circular RNAs (circRNAs) are emerging as crucial regulators in tumorigenesis and aggressive progression. However, their biological roles in non-small cell lung cancer (NSCLC) remain largely unknown. Here, by performing circRNA high throughput sequencing in 4 paired NSCLC and normal tissues, we found a NSCLC-associated circRNA, circ-PLCD1, which was evidently downregulated in NSCLC tissues and cell lines. Circ-PLCD1 was transcriptionally activated by tumor-inhibiting protein p53, and exogenous expression of circ-PLCD1 inhibited NSCLC cell proliferation, invasion and induced apoptosis. Mechanistically, circ-PLCD1 acted as a competitive endogenous RNA (ceRNA) to sponge miR-375 and miR-1179 and elevate PTEN, a well-known inhibitor of oncogenic PI3K/AKT signaling, thereby repressing NSCLC tumorigenesis. Importantly, we also identified this ceRNA regulatory axis of circ-PLCD1/miR-375/miR-1179/PTEN in vivo by establishing a xenograft tumor model. Clinically, NSCLC patients with low circ-PLCD1 expression had larger tumor size, later clinical stage and shorter survival time than those with high circ-PLCD1 expression. Altogether, our findings reveal the important tumor suppressive role of circ-PLCD1 in NSCLC, reactivation of this circRNA may be considered as a novel therapeutic avenue for patient with NSCLC.

Significance of screening sensitive methylation sites using whole-genome sequencing in early diagnosis of non-small cell lung cancer.

The current study aimed to screen the sensitive methylation sites of non-small cell lung cancer by whole-genome sequencing and construct an early warning system for lung cancer. For this purpose, from June 2017 to December 2020, fresh NSCLC tissues and paired adjacent NSCLC tissues from 45 patients were collected. DNA and total RNA were extracted from non-small cell lung cancer (NSCLC) and paired non-cancerous lung tissues. The DNA library combined with a biotinylated probe was collected by Dynabeads m270 streptavidin beads. The concentration of the final library was determined by qubit dsDNA HS assay. Quantitative analysis of DMR methylation in 45 paired tumor and normal lung tissues was performed. RT qPCR and Western blot were used to verify the mRNA expression of candidate genes. Results showed that the methylation rate of CpG 7 in stxbp6 in stage III NSCLC was higher than that in stage I and early-stage II NSCLC; The methylation rates of cpg1 and 38-39 units in fzd10 were higher in stage I NSCLC than in stage II and III NSCLC; The methylation rates of CpG 6 in stxbp6 and CpG 4 and 20-21 in bcl6b in patients with tumor diameter > 3cm were higher than those in patients with tumor diameter < 3cm; Methylation of CpG unit 3 in stxbp6 is associated with age. Stxbp6, bcl6b, fzd10 and hspb6 mRNA expression were down-regulated in patients under 45 years old. The methylation rates of CpG 7 in stxbp6, CPG 6 in stxbp6 and CpG 4 and 20-21 in bcl6b were negatively correlated with the survival time of patients; The methylation rates of CpG 1 and 38-39 units in fzd10 were positively correlated with survival time (P<0.05). It was concluded that the methylation rates of CpG 7 in stxbp6, CPG 6 in stxbp6 and CpG 4 and 20-21 in bcl6b are valuable for early diagnosis.

Correlation of SIDT1 with Poor Prognosis and Immune Infiltration in Patients with Non-Small Cell Lung Cancer.

PurposeTo investigate the expression profile of systemic RNA interference deficient-1 transmembrane family member 1 (SIDT1) in patients with non-small cell lung cancer (NSCLC) and assess its prognostic value and immune infiltration.Materials and MethodsA tissue microarray (TMA) was purchased containing 60 paired NSCLC tissues and matched para-tumor tissues, and another TMA was constructed comprising 140 NSCLC tissues. Then, immunohistochemical staining and scoring were performed. Finally, we evaluated the expression profiles and prognostic values of SIDT1 in NSCLC. Correlation between SIDT1 gene expression and the immune microenvironment of NSCLC was investigated using the ESTIMATE and CIBERSORT algorithms.ResultsSIDT1 was mainly detected in the cytoplasm and the cell membrane. SIDT1 expression was significantly higher in tumor tissues than in para-tumor tissues. High expression of SIDT1 was correlated with the NSCLC stage. High SIDT1 expression signals worsening overall survival (OS) in NSCLC patients, especially in stage I. In LUSC, high SIDT1 expression is an independent prognostic factor for worsening OS.ConclusionHigh SIDT1 expression predicted a low survival rate in LUSC patients, suggesting it may be a potential target for prognostic assessment of NSCLC patients.

MYPT1, regulated by miR-19b-3p inhibits the progression of non-small cell lung cancer via inhibiting the activation of wnt/β-catenin signaling

AimsMyosin phosphatase targeting protein 1 (MYPT1) was identified to function as a tumor suppressor in several kinds of cancers, but its role and the molecular mechanisms in non-small cell lung cancer (NSCLC) remain undiscovered. Herein, we aimed to reveal MYPT1 expression pattern and role in NSCLC, and investigate the underlying mechanisms. Main methodsSixty-eight paired NSCLC tissues and the adjacent normal tissues were included in this study. Western blotting and quantitative reverse transcription-polymerase chain (qPCR) technologies were applied for protein and RNA detection. CCK-8, colony formation, flow cytometry, wound healing, transwell chambers coated with Matrigel and in vivo experiments were applied to detect cell viability, colony formation, apoptosis, migration, invasiveness and tumorigenesis, respectively. Key findingsMYPT1 expressed at a lower level in NSCLC tissues as compared with the adjacent normal tissues, which predicted advanced clinic process and poor prognosis. Overexpression of MYPT1 resulted in obvious inhibitions in cell viability, colony formation, migration, invasiveness and tumorigenesis, and induced cell apoptotic rates, as well as decreased the expression levels of β-catenin and TCF4. Besides, overexpression of β-catenin weakened the above roles of MYPT1. In addition, the luciferase gene reporter assay verified that MYPT1 was a target of miR-19b-3p. Further experiments showed that miR-19b-3p promoted cell viability, invasiveness and migration and repressed cell apoptosis by targeting MYPT1. SignificanceIn conclusion, this study demonstrates that MYPT1, regulated by miR-19b-3p, inhibits the progression of NSCLC via inhibiting the activation of wnt/β-catenin signaling.

Comprehensive analysis of prognostic value and immune infiltration of kindlin family members in non-small cell lung cancer

BackgroundKindlin Family Members have been reported to be aberrantly expressed in various human cancer types and involved in tumorigenesis, tumor progression, and chemoresistance. However, their roles in non-small cell lung cancer (NSCLC) remain poorly elucidated.MethodsWe analyzed the prognostic value and immune infiltration of Kindlins in NSCLC through Oncomine, GEPIA, UALCAN, CCLE, Kaplan‑Meier plotter, cBioPortal, TIMER, GeneMANIA, STRING, and DAVID database. Additionally, the mRNA expression levels of Kindlins were verified in 30 paired NSCLC tissues and NSCLC cell lines by real-time PCR.ResultsThe expression level of FERMT1 was remarkably increased in NSCLC tissues and NSCLC cell lines, while FERMT2 and FERMT3 were reduced. Kindlins expressions were associated with individual cancer stages and nodal metastasis. We also found that higher expression level of FERMT1 was obviously correlated with worse overall survival (OS) in patients with NSCLC, while higher FERMT2 was strongly associated with better overall survival (OS) and first progression (FP). Additionally, the expression of FERMT2 and FERMT3 were obviously correlated with the immune infiltration of diverse immune cells. Functional enrichment analysis has shown that Kindlins may be significantly correlated with intracellular signal transduction, ATP binding and the PI3K-Akt signaling pathway in NSCLC.ConclusionsThe research provides a new perspective on the distinct roles of Kindlins in NSCLC and likely has important implications for future novel biomarkers and therapeutic targets in NSCLC.

High KIF11 expression is associated with poor outcome of NSCLC.

To clarify the correlation between KIF11 (kinesin family member 11) and clinicopathologic characteristics of non-small cell lung cancer (NSCLC) and identify the prognostic value of KIF11 in patients with NSCLC. For investigating the expression of KIF11 in NSCLC, two tissue microarrays (TMAs: one contained 60 paired NSCLC tissues and paratumor tissues, the other contained 140 NSCLC tissues and 10 normal lung tissues) were constructed, stained, and scored. The Cancer Genome Atlas (TCGA) datasets were used to explore the differential expression level of KIF11 between NSCLC and paratumor. Kaplan-Meier survival curves were plotted and multivariate analysis were carried out. The staining of KIF11 mainly distributed throughout the cytoplasm of tumor cells. Its expression was higher in NSCLC than paratumor cells, and similar results were obtained from TCGA datasets. We found that high expression of KIF11 had a significant correlation with lymph node metastases (p = 0.024) and pathologic stage (p = 0.018); that significant difference was not found in any other clinicopathologic characteristic. As univariate and multivariate analysis showed, KIF11 expression was significantly correlated with overall survival time of NSCLC (p = 0.002, p = 0.025, respectively). High KIF11 expression was found to significantly associate with overall survival of stage II-III (p = 0.001) and lung adenocarcinoma (p = 0.036). High KIF11 expression predicts poor outcome in NSCLC. KIF11 is expected to be a viable prognostic biomarker for NSCLC.

MiR‑7/SP1/TP53BP1 axis may play a pivotal role in NSCLC radiosensitivity.

MicroRNA-7 (miR-7) has been identified as a tumor suppressor in non-small cell lung cancer (NSCLC) and a radiosensitivity regulator. Numerous studies have revealed that specific protein 1 (SP1) plays a critical role in the tumorigenesis of various types of cancers and regulates radiosensitivity and tumor suppressor p53-binding protein 1 (TP53BP1), which plays an essential role in DNA repair. However, it is not clear whether miR-7 has a regulatory effect on SP1 and TP53BP1 in NSCLC. In the present study it was revealed that miR-7 directly binds to the 3′UTR of SP1, thereby suppressing SP1 expression to regulate radiosensitivity. Overexpression of miR-7 and SP1 and knockdown of miR-7 and SP1 were performed using lentiviral transfection. Protein and mRNA abundance of SP1 and TP53BP1 were determined using western blotting and RT-qPCR, respectively, while miR-7 binding to SP1 was validated using a luciferase reporter assay. Biological function analysis indicated that miR-7 negatively regulated SP1 and inhibited cell proliferation, migration, and invasion when combined with radiation. It was also revealed that the expression of TP53BP1 was positively regulated by SP1 or negatively regulated by miR-7. In conclusion, SP1 was a target of miR-7, and the decreased expression of SP1 resulting from miR-7 overexpression in NSCLC was vital for improving radiosensitivity in NSCLC cells. Moreover, SP1 expression was detected in 95 paired NSCLC and adjacent normal tissues, and it was determined that SP1 was significantly upregulated in NSCLC tissues and that its upregulation was correlated with the degree of tissue differentiation. Thus, SP1 and/or miR-7 may be potential molecular targets in NSCLC radiotherapy.

Effects of miR-218-1-3p and miR-149 on proliferation and apoptosis of non-small cell lung cancer cells.

The aim of the present study was to explore the effects of miR-218-1-3p and miR-149 on the biological function of non-small cell lung cancer (NSCLC) cells A549. Paired NSCLC and adjacent tissues were obtained from 50 NSCLC patients admitted to Shandong Provincial Chest Hospital Affiliated to Shandong University (Jinan, China) from April 2015 to May 2018. The expression levels of miR-218-1-3p and miR-149 were detected by reverse transcription-quantitative PCR (RT-qPCR). The lung adenocarcinoma A549 cells were assigned into the blank group (without transfection), negative control (NC) group (transfected with miRNA NC), and the transfected groups miR-218-1-3p mimic and miR-149 mimic groups. Proliferation and cell growth were determined by CCK-8 assay and cell invasion ability in vitro was assessed by Transwell assay. Flow cytometry was carried out for the detection of cell apoptosis. RT-qPCR results showed that the expression levels of miR-218-1-3p and miR-149 in NSCLC tissues were significantly lower than those in adjacent tissues (P<0.001). At 48 and 72 h, the cell growth of the A549 cells in the miR-218-1-3p mimic and miR-149 mimic groups was significantly lower than that in the NC and blank groups (P<0.05). The number of invasive cells in the miR-218-1-3p mimic and miR-149 mimic groups was significantly lower than that in the NC and blank groups (P<0.05). The apoptotic rate of A549 cells in the miR-218-1-3p mimic and miR-149 mimic groups was significantly higher than that in the NC and blank groups (P<0.05). In conclusion, upregulation of miR-218-1-3p and miR-149 can inhibit the proliferation, invasion and migration of A549 cells in NSCLC, thereby promoting the apoptosis of A549 cells. Thus, miR-218-1-3p and miR-149 can be used as new molecular targets for the diagnosis and treatment of NSCLC.

LncRNA SNHG9 is Downregulated in Non-Small Cell Lung Cancer and Suppressed miR-21 Through Methylation to Promote Cell Proliferation.

BackgroundLncRNA SNHG9 has been shown to be an oncogenic lncRNA in glioblastoma, while its role in other cancers is unknown. The aim of this study was to investigate the role of SNHG9 in non-small cell lung cancer (NSCLC).MethodsThe differential expression of SNHG9 in NSCLC was first explored by analyzing the TCGA dataset, followed by measuring the expression levels of SNHG9 in paired NSCLC and non-tumor tissues by RT-qPCR. Expression of miR-21 was also determined by RT-qPCR. Correlations were analyzed by linear regression. The interaction between miR-21 and SNHG9 was detected using RNA pull-down. The expression relationship between SNHG9 and miR-21 was analyzed by SNHG9 or miR-21 overexpression experiments. The effects of overexpression of SNHG9 on the methylation of miR-21 were analyzed by methylation-specific PCR (MSP). Cell proliferation was evaluated by CCK-8 assay.ResultsBy analyzing the TCGA dataset, we observed downregulation of SNHG9 in NSCLC, which was confirmed by measuring the expression levels of SNHG9 in paired NSCLC tumor tissues and non-tumor tissues from NSCLC patients involved in this study. MiR-21 was upregulated in NSCLC tumor tissues and inversely correlated with SNHG9 in cancer tissues but not in non-tumor tissues. The interaction between SNHG9 and miR-21 was predicted by bioinformatic analyses, which was further verified by RNA pull-down. In NSCLC cells, overexpression of SNHG9 led to downregulated miR-21 and increased methylation of miR-21 gene. In contrast, miR-21 did not affect the expression of SNHG9. In addition, overexpression of SNHG9 attenuated the enhancing effects of miR-21 on NSCLC proliferation.ConclusionSNHG9 might downregulate miR-21 through methylation to suppress cancer cell proliferation.

PINK1 Overexpression Promotes Cell Migration and Proliferation via Regulation of Autophagy and Predicts a Poor Prognosis in Lung Cancer Cases.

BackgroundLung cancer remains the leading cause of cancer-related death worldwide. The human PINK1 gene (PTEN induced kinase 1, Park6), an important gene for Parkinson’s disease, was found to be associated with tumor development although the molecular mechanisms underlying this relationship remain largely unknown.ObjectiveTo analyze the clinical value and molecular mechanism of PINK1 in non-small cell lung cancer (NSCLC).Materials and MethodsWestern blot, qRT-PCR and Immunohistochemistry were employed to determine the levels of PINK1 in 87 paired NSCLC tissues, Oncomine and TCGA databases were also used for the evaluation of expression and prognosis of PINK1. The mitophagy, proliferation, migration, invasion, and apoptosis abilities of A549 and H1975 cells were detected, and the autophagy-related proteins in the cells were also determined.ResultsImmunohistochemical staining revealed higher PINK1 expression in tumor tissues, which was strongly linked to the tumor-node-metastasis classification. Survival analysis of 1085 NSCLC patients also revealed that low PINK1 expression levels were associated with significantly longer overall survival. Univariate and multivariate analyses indicated that PINK1 expression was an independent predictor of overall survival among patients with NSCLC. We also evaluated the influence of PINK1 deficiency in NSCLC cell lines (A549 and H1975), which revealed significant suppression of migration capability and cell viability, as well as a significantly elevated apoptosis ratio. In cells with stable interference of PINK1 expression, dysfunctional mitochondria accumulated while autophagy was inhibited, which indicated that cell activity suppression was mediated by the accumulation of dysfunctional mitochondria. The suppression of migration and autophagy was reversed in cells that overexpressed PINK1.ConclusionOur results suggest that PINK1 may be a potential therapeutic target and prognostic biomarker in NSCLC.

Endogenous thrombopoietin promotes non-small-cell lung carcinoma cell proliferation and migration by regulating EGFR signalling.

Thrombopoietin (TPO) is a haematopoietic cytokine mainly produced by the liver and kidneys, which stimulates the production and maturation of megakaryocytes. In the past decade, numerous studies have investigated the effects of TPO outside the haematopoietic system; however, the role of TPO in the progression of solid cancer, particularly lung cancer, has not been well studied. Exogenous TPO does not affect non‐small‐cell lung cancer (NSCLC) cells as these cells show no or extremely low TPO receptor expression; therefore, in this study, we focused on endogenous TPO produced by NSCLC cells. Immunohistochemical analysis of 150 paired NSCLC and adjacent normal tissues indicated that TPO was highly expressed in NSCLC tissues and correlated with clinicopathological parameters including differentiation, P‐TNM stage, lymph node metastasis and tumour size. Suppressing endogenous TPO by small interfering RNA inhibited the proliferation and migration of NSCLC cells. Moreover, TPO interacted with the EGFR protein and delayed ligand‐induced EGFR degradation, thus enhancing EGFR signalling. Notably, overexpressing TPO in EGF‐stimulated NSCLC cells facilitated cell proliferation and migration, whereas no obvious changes were observed without EGF stimulation. Our results suggest that endogenous TPO promotes tumorigenicity of NSCLC via regulating EGFR signalling and thus could be a therapeutic target for treating NSCLC.

IL-6 stimulates lncRNA ZEB2-AS1 to aggravate the progression of non-small cell lung cancer through activating STAT1.

To illustrate the role of interleukin 6 (IL-6) in the progression of non-small cell lung cancer (NSCLC) via activating STAT1. The level of IL-6 mRNA in 48 paired NSCLC tissues and matched normal ones was determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Kaplan-Meier curves were depicted for assessing the overall survival of NSCLC patients with high or low level of IL-6 mRNA. Subsequently, the ZEB2-AS1 level in A549 cells treated with different doses of IL-6 for different time points was determined. After A549 cells were treated with different doses of IL-6, wound closure assays were performed to assess the migration of cells. Protein levels of pSTAT1 and STAT1 in IL-6-treated A549 cells were detected by Western blot. The regulatory effect of STAT1 on IL-6-induced migration of A549 cells was also evaluated. The interaction between ZEB2-AS1 and STAT1 was explored through Chromatin Immunoprecipitation (ChIP) assay. Finally, the role of ZEB2-AS1/STAT1 axis in regulating NSCLC cells was investigated through rescue experiments. Our results indicated that IL-6 was upregulated in NSCLC tissues and cancer cell lines. NSCLC patients with T3-T4 or accompanied with lymphatic metastasis had a higher IL-6 abundance than those with T1-T2 or without metastatic foci. The worse prognosis was identified in NSCLC patients with high expression of IL-6 compared to those with low expression. ZEB2-AS1 showed dose-dependent and time-dependent increase in IL-6-treated A549 cells. IL-6 treatment gradually enhanced the migration ability of A549 cells in a dose-dependent manner. In IL-6-treated A549 cells, protein level of pSTAT1 was remarkably upregulated, and knockdown of STAT1 significantly reversed the promotive effect of IL-6 on migration ability of A549 cells. The results of ChIP assay verified the interaction between ZEB2-AS1 and STAT1. In addition, ZEB2-AS1 could reverse the regulatory effect of STAT1 on the migration ability of A549 cells. IL-6 was upregulated in NSCLC and accelerated the progression of NSCLC via activating STAT1/ ZEB2-AS1.

High expression of RAD21 predicts poor survival in patients with operated non-small-cell lung cancer.

To investigate the expression profile and prognostic value of RAD21 in patients with non-small cell lung cancer (NSCLC). A tissue microarray (TMA) containing 60 paired NSCLC tissues and peritumor tissues was purchased and another TMA containing 140 NSCLC tissues was constructed. Then, immunohistochemical staining was performed and scored. Finally, the expression profile and prognostic value of RAD21 were evaluated. RAD21 was predominantly detected in the nucleus of tumor and peritumor cells. RAD21 was more highly expressed in tumor tissues compared to peritumor tissues. High RAD21 expression was correlated with more lymph node metastases and advanced pathological stage, but not with any other clinicopathological features. High RAD21 expression led to worsened overall survival (OS) and was an independent prognostic factor for worsened OS in NSCLC, especially in stage II-III. High RAD21 expression indicates poor survival in patients with NSCLC. RAD21 may become a novel prognostic biomarker and therapeutic target in patients with NSCLC.

MicroRNA-424-5p inhibits the development of non-small cell LCa by binding to ITGB1.

The aim of this study was to explore the effect of microRNA-424-5p on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) cells, and to investigate its influence on the expression of ITGB1 and potential regulatory mechanism. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the level of microRNA-424-5p in 44 paired NSCLC tissues and adjacent tissues. The relation between microRNA-424-5p expression and NSCLC clinical indicators was analyzed. Subsequently, microRNA-424-5p mimics and inhibitors were transfected into NSCLC cells to construct microRNA-424-5p overexpression or knockdown models, respectively. QRT-PCR was used to further verify the transfection efficiency. A series of experiments, including cell counting kit-8 (CCK-8) assay, colony formation, 5-Ethynyl-2'-deoxyuridine (EdU), and flow cytometry were used to analyze the effect of microRNA-424-5p on the biological function of NSCLC A549 and H358 cells. Finally, the potential association between microRNA-424-5p and its downstream gene ITGB1 was explored through luciferase reporter gene assay and cell recovery experiment. QRT-PCR results showed that microRNA-424-5p level was significantly lower in NSCLC tissues than that of adjacent normal tissues. Compared with patients with high expression of microRNA-424-5p, the pathological stage of those with low expression of microRNA-424-5p was significantly higher. In vitro experiments showed that microRNA-424-5p overexpression remarkably decreased cell proliferation and increased cell apoptosis, which were further validated in microRNA-424-5p inhibitor group. Subsequently, ITGB1 expression was found significantly up-regulated in NSCLC cell lines and tissues. Meanwhile, ITGB1 expression was negatively correlated with microRNA-424-5p level. In addition, a recovery experiment indicated that overexpression of ITGB1 could counteract the effect of microRNA-424-5p mimics on the proliferation and apoptosis of NSCLC cells. All these findings revealed that microRNA-424-5p and ITGB1 affected the malignant progression of NSCLC. MicroRNA-424-5p was closely correlated with the pathological stage and poor prognosis of NSCLC, thereby inhibiting the occurrence and development of NSCLC.